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Environmental Research

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Effects of mobile phone exposure on metabolomics in the male and femalereproductive systems

Gamze Altuna, Ömür Gülsüm Deniza, Kıymet Kübra Yurta,c, Devra Davisb,c, Süleyman Kaplana,⁎

a Department of Histology and Embryology, Faculty of Medicine, Ondokuz Mayıs University, Samsun, TurkeybHadassah Medical School, Hebrew University, Jerusalem, Isreal and Faculty of Medicine, Ondokuz Mayıs University, Samsun, Turkeyc Environmental Health Trust, 7100 N Rachel Way Unit 6 Eagles Rest, Teton Village, WY 83025, United States

A R T I C L E I N F O

Keywords:Mobile phone exposureOxidative stressSpermatogenesisOogenesis

A B S T R A C T

With current advances in technology, a number of epidemiological and experimental studies have reported abroad range of adverse effects of electromagnetic fields (EMF) on human health. Multiple cellular mechanismshave been proposed as direct causes or contributors to these biological effects. EMF-induced alterations incellular levels can activate voltage-gated calcium channels and lead to the formation of free radicals, proteinmisfolding and DNA damage. Because rapidly dividing germ cells go through meiosis and mitosis, they are moresensitive to EMF in contrast to other slower-growing cell types. In this review, possible mechanistic pathways ofthe effects of EMF exposure on fertilization, oogenesis and spermatogenesis are discussed. In addition, thepresent review also evaluates metabolomic effects of GSM-modulated EMFs on the male and female reproductivesystems in recent human and animal studies. In this context, experimental and epidemiological studies whichexamine the impact of mobile phone radiation on the processes of oogenesis and spermatogenesis are examinedin line with current approaches.

1. Introduction to metabolomics: effects of EMF on male andfemale reproduction

Humans are often exposed to a broad array of electromagnetic fields(EMF) as technology advances. The purpose of this review is to discussthe effects of EMF-induced metabolomics on the male and female re-productive systems caused by mobile phones EMF exposure in the lightof current research. Human and animal studies have identified anumber of adverse health effects that can be caused by this exposurethat require additional evaluations (Sepehrimanesh and Davis, 2017).Many factors, such as body weight, dielectric constant, electrolytebalance and other properties are important in the context of these ef-fects since they change conductivity and reactivity to EMF (Tabrahet al., 1998; Vesselinova, 2015). In vivo and in vitro studies indicate thatEMF exposure can alter cellular homeostasis, activate voltage-gatedcalcium channels and affect endocrine function, reproduction functionsand fetal growth in animals. Moreover, the absorption rate of EMF inhuman cells is affected by polarization, amplitude, frequency, andpower density. Absorption rates of different tissues affect cellular ab-sorption of radiofrequency energy. The effects of EMF may be classifiedas thermal or non-thermal. In general, the thermal mechanism is un-derstood to increase local temperature with an oscillatory current at the

radio frequency of the electrical field (Belyaev, 2005; Gye and Park,2012). In contrast, non-thermal effects of EMF on human cells can in-clude the generation of large and damaging reactive oxygen species(ROS). ROS generation causes oxidative damage in the target cells. Inresearch on pregnant rats and their offspring exposed to 900MHz,1800MHz and 2450MHz EMF, Yuksel et al. (2016) reported a higherbody temperature and uterus lipid peroxidation levels in newborn ratscompared to a control group. This indicates that health risks caused byEMF can arise from either non-thermal or thermal effects of EMF in thefemale reproductive system (Yuksel et al., 2016) and that both may beinvolved. EMF has a high penetrating power and can accelerate themovement of charged particles of macromolecules and polymers, suchas electrons and ions (Asghari et al., 2016).

EMF-induced changes observed at the cellular level may cause freeradicals and Ca+2 mediated cell growth inhibition, protein misfoldingand DNA damage. EMF has biochemical interactions arising from ac-tivation of second chemical messengers in biological materials(Belyaev, 2005; Gye and Park, 2012). Moreover, the effect of EMF ex-posure on reproductive functions depends on frequency and wave,polarity and information content, as well as energy, power density andtotal time of exposure. (Gye and Park, 2012). The main side-effects ofEMF are seen through protein synthesis, which seriously affects cell

https://doi.org/10.1016/j.envres.2018.02.031Received 28 September 2017; Accepted 22 February 2018

⁎ Correspondence to: Department of Histology and Embryology, Medical Faculty, Ondokuz Mayıs University, 55139 Samsun, Turkey.E-mail address: skaplan@omu.edu.tr (S. Kaplan).

Environmental Research 167 (2018) 700–707

Available online 05 June 20180013-9351/ © 2018 Elsevier Inc. All rights reserved.

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structure and functions (Mancinelli et al., 2004). In that context, Desaiet al. suggested that the EMF might enhance ROS generation by in-creasing nicotinamide adenine dinucleotide (NADH) oxidase activity inplasma membrane. One possible interaction mechanism between elec-tromagnetic radiation and biological systems is a process involving freeradicals (Desai et al., 2009b). Highly reactive free radical formationincreases oxidative stress, a physiological or cellular condition, andmolecular damage subsequently occurs, together with pronounced ef-fects on macromolecules such as. ROS consist of highly reactive mole-cules formed by unconjugated electrons in free orbits. While a smallconcentration of ROS is essential for the maintenance of healthy cells,excessive concentrations can cause tissue damage and impair the abilityof cells to undergo repair or apoptosis (Bandyopadhyay et al., 1999;Kesari et al., 2013). In the case of exposure to mobile phone radiation,an EMF-induced iron-mediated process (Fenton reaction) occurs. Thisreaction leads to an increase in hydroxyl free radical generation in thecells that can cause serious damage. Hydroxyl radicals are generatedfrom hydrogen peroxide through the Fenton reaction in the presence ofiron, and also EMF (D'Autreaux and Toledano, 2007; Desai et al.,2009a; Kesari et al., 2013). Kesari et al. (2013) suggested that theFenton reaction induced by EMF accounts directly for cell death byincreasing the production of free radicals that play an important role inthe enzymatic and genetic action of EMF (Fig. 1) (Kesari et al., 2013).

EMF-induced free radicals can create both structural deformitiesand functional defects in sperm (Gye and Park, 2012). Processes re-levant to this damage can involve the stimulation of protein kinases,and an increase in cyclic adenosine mono phosphate (cAMP), phos-phorylation, and calcium efflux. The first target of ROS is the spermmembrane. Lipid peroxidation has been shown to be induced when O2

is not soluble in the hydrophobic membrane or when it is in the pro-tonated form (Evans and Halliwell, 1999; Phillips et al., 2009). More-over, Kumar et al. (2011) reported that exposure to 2.45-GHz EMFcaused apoptosis during spermiogenesis, and that caspase-3 activityaffects reproductive physiology. The effect of oxidative stress on healthand of microwave EMFs on chronic stress through over-production ofROS have now been revealed (Kumar et al., 2011). Excessive generationof ROS and oxidative stress may contribute to aging and various healthconditions affecting female reproduction. Endothelial dysfunctioncaused by oxidative stress contributes to obstetric complications, suchas early and repeated pregnancy loss, pre-eclampsia, intrauterinegrowth restriction and preterm delivery (Webster et al., 2008). Reactiveoxygen and nitrogen species may have adverse effects on embryo im-plantation and may affect the development of reproductive disorders,such as endometriosis and pre-eclampsia (Webster et al., 2008). Despitethe fact that the exact pathogenesis of pre-eclampsia is still unknown,placental ischemia/hypoxia is regarded as a significant factor through

the induction of oxidative stress, that may trigger endothelial celldysfunction in the disease (Webster et al., 2008; Possomato-VieiraandKhali, 2016). Modified vasomotor functions are demonstrated withunsuccessful embryo implantation in preeclampsia and endometriosis,and low placental perfusion (Massé et al., 2002). Antioxidants are re-ported to cure such effects and can thus mitigate infertility risks(Agarwal et al., 2008b; Webster et al., 2008). Previous studies haveexamined the deleterious effects of EMF on reproduction in both gen-ders in different exposure doses, and research has shown that EMF hasharmful effects on sex hormones, gonadal function, fetal growth andpregnancy (Rodriguez et al., 2003; Rodriguez et al., 2004; Gye andPark, 2012). In addition, natural antioxidants can be used to reduce theside-effects of EMF exposure (Gye and Park, 2012; Nelson et al., 1995;Pourlis, 2009). In this regard, ROS over-generation can lead to dis-ruption of normal female physiological reactions by penetrating thebody's natural antioxidant defense system (Agarwal et al., 2012; Al-Gubory et al., 2010). Oxidative stress has been shown to be a significantcause of infertility in men. Sperm numbers and motility are importantdeterminants of fertilization capacity, and oxidative stress may causeinfertility in men by affecting these parameters (Makker et al., 2009;Saalu, 2010).

A significant increase has been observed in mitochondrial and cy-tosolic superoxide generation in human spermatozoa exposed to EMF(Agarwal et al., 2009; De Iuliis et al., 2009). The relation between lossof sperm motility and ROS over-generation is well-known in spermbiology (Darr et al., 2016). Increases in lipid peroxidation and the se-quent malondialdehyde depend on acrolein generation that can cova-lently bind with electrophilic aldehyde and proteins such as 4-hyr-doxinonena (4-HNE) (Aitken et al., 2012). These components alkalizethe sperm axonemal proteins that regulate sperm motility, and espe-cially the heavy chain dynein. In addition, electrophiles such as 4-HNEincrease oxidative stress by stimulating ROS generation through spermmitochondria (Aitken et al., 2012). This occurs since the mitochondrialelectron transport chain, another protein group alkalized by 4-HNE,generates succinic acid dehydrogenase components (Aitken et al.,2012). Even a minimal increase in ROS induced with EMF can have aprofound effect on reproduction by damaging mitochondria (Aitkenet al., 2012). ROS generation originating from EMF is reported to in-crease lipid peroxidation in spermatozoa and damage mitochondrialDNA (Aitken et al., 2012; Al-Damegh, 2012; Kesari et al., 2011;Moazamian et al., 2015).

2. The effects of EMF-induced oxidative stress mechanisms on themale genital system

The effects of EMF emitted from the mobile phone on the re-productive organs and fetal development have been extensively in-vestigated by our laboratories (Sepehrimanesh et al., 2014) and others,and the number of studies on the impact of EMF exposure on humanreproduction has increased considerably. While some studies suggestthat EMF has an adverse effect on male reproductive systems, othershave reported that EMF has no, or only partial, effects on testiculartissue and functions. As with other endpoints, these inconsistent resultsmay be due to the fact that different researchers have used differentfrequencies, amplitudes, power densities, and that the density and ex-posure times of the induced magnetic fields have also not been stan-dardized (Belyaev, 2005; Dasdag et al., 2003; Desai et al., 2009b; Yanet al., 2007). Radiation emitted by mobile phones may cause structuraland functional injury in the testes, changes in semen parameters, anddecreased epididymal sperm concentrations and male fertility (Kanget al., 2010). These changes depend on the length of exposure, thespecific absorption rate (SAR) and the energy level of EMF. In thiscontext, a decrease in testicular size has been reported as one effect, andstudies have also reported decreases in the diameter and epithelialthickness of the seminiferous tubules (Dasdag et al., 1999; Ozguneret al., 2005; Salama et al., 2010). According to De Iuliis et al. (2009),

Fig. 1. Diagram showing the role of Fenton reaction EMF-induced cellular damage(modified from Phillips et al., 2009).

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the underlying mechanisms involved in cellular damage to the malereproductive system caused by EMF are unclear. However, sperm cellsare particularly vulnerable to oxidative damage due to the absence ofantioxidant enzymes as well as substrates to protect against free radicalattack on their surfaces (De Iuliis et al., 2009). Another study of malerats reported that mobile phone exposure increased glutathione levels.In addition, exposure to 900 and 1800MHz GSM mobile phones (1 hper day for 28 days) has been demonstrated to reduce glutathioneconcentrations and to increase lipid peroxidation in male rats(Mailankot et al., 2009). The rise in ROS production resulting fromexposure to 0.9W/kg EMF for 35 h (2 h/day) in standby mode increasesprotein kinase activity. Additionally, the EMF emitted by mobilephones has also been reported to adversely impact the fertilizationcapacity of spermatozoa (Kesari et al., 2010, 2011). Esmekaya et al.(2011) determined that exposure to 900MHz EMF (1.20W/kg and20min/day for 3 weeks) causes oxidative damage in testicular tissue byenhancing nitric oxide and inhibiting antioxidant mechanisms(Esmekaya et al., 2011).

2.1. Mobile phone and sperm motility

Many studies have investigated the effects of EMF on the male re-productive system (Gul et al., 2009; Odaci and Ozyilmaz, 2015; Odaciet al., 2016; Sepehrimanesh et al., 2014). While some authors believethat the both the thermal and non-thermal effects emitted by mobilephones are deleterious to health, others have concluded that the non-thermal effects are minimal. (Black and Heynick, 2003; Jauchem, 2003;Meltz, 2003). In this context, studies have clearly shown that tem-perature increases caused by mobile phones can adversely affect spermmaturity and motility (Fig. 2) (Bonde et al., 1996; Brusick et al., 1998;Fejes et al., 2005).

Oxidative stress is also considered an important cause of maleinfertility. In this context, ROS, which originate from spermatozoa andleukocytes, affect sperm motility and can cause infertility (Figs. 2 and3). The plasma membrane of spermatozoa has a multiple redox systemwhich resembles NADH, an important source of superoxide anions.Studies have shown that EMF stimulates NADH oxidase in the plasmamembrane of mammalian cells (Maneesh et al., 2005a, 2005b).

ROS are maintained at physiologically low levels by intracellularfree radical scavengers. Glutathione (GSH), an important scavenger inliving organisms, plays a critical role in the body's antioxidant defensemechanisms against free radicals. Conditions disrupting intracellularGSH levels cause significant changes in cellular metabolism. GSH in thetissues is a determinant of their detoxification capacity, cell redoxbalance and cell protection. A study suggested an increase in GSHconsumption in human ejaculate exposed to RF-RM, and posited thatthe ROS generated after exposure might be responsible for a low level ofsperm (Salama et al., 2010). In brief, an increase in the oxidative stresscaused by RF-EMF leads to a decrease in testicular function (Agarwalet al., 2009; Irvine, 1996; Salama et al., 2010; Sepehrimanesh andDavis, 2017). In a study based on a similar hypothesis, Mailankot et al.reported a low GSH concentration in the testes and epididymis, and anincreased lipid concentration in 10–12-week Wistar albino rats exposedto 900 and 1800MHz EMF over 28 days (1 h per day) (Mailankot et al.,2009). An increased level of free radicals in the cells may be induced bylipid peroxidation through oxidative degradation of polyunsaturated fatacids in the cell membranes. Damage to the structure of the lipid matrixof the spermatozoa membrane and axoneme then occurs through lipidperoxidation of sperm. However, rapid loss of intracellular ATP is re-lated to a decrease in sperm viability and increased structural defects,and spermatogenesis can thus be inhibited (Ceribasi et al., 2012; Turket al., 2008).

Recent studies have investigated the potential effects of EMF onsperm quality (Fejes et al., 2005; Kilgallon and Simmons, 2005; Turket al., 2008). In this context, Kilgallon et al. suggested that keepingmobile phones close to the testicles has a significant adverse effect onconcentrations and percentages of mobile sperm (Kilgallon and

Fig. 2. Exposure to RF-EMF causes an alteration in calcium efflux and a decrease in theactivity of protein kinase. The figure shows a schematic representation of biological ef-fects associated with male infertility. hsp: heat shock protein, bFGF: basic fibroblastgrowth factor, MAP: mitogen-activated protein (adapted from Desai et al., 2009b;Hamada et al., 2011).

Fig. 3. Potential effects of EMF emitted from mobile phones on the association betweenthe central nervous system and the reproductive system and the role of EMF on maleinfertility (Kesari et al., 2013).

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Simmons, 2005). In addition, a study of 271 healthy males by Fejeset al. (2005) demonstrated no significant effect of communication sig-nals in standby mode on spermatozoa. The authors therefore concludedthat long-term mobile phone use may affect sperm motility. Althoughthe general motility of sperm did not change, the moderate decreaseobserved in grade A motility and the moderate increase observed ingrade B motility may be a result of the EMF emitted from mobile phones(Fejes et al., 2005). Additionally, Sun et al. (2005) demonstrated thatthe EMF emitted from computers did not alter sperm quality (Sun et al.,2005). Erogul et al. (2006) reported that exposure to EMF causes adecrease in sperm motility. Semen analysis in the exposure group andcontrol group showed significant alterations in sperm motility. (Erogulet al., 2006). Similarly, Imai et al. (2011) examined the effects of EMFon offspring male rats. No differences were determined in terms of testisweight between the control group and rats exposed to 1.95 GHz (SAR0.4–0.08W/kg). In addition, no abnormalities were observed in termsof sperm motility and morphology or the histological appearance ofseminiferous tubules (Imai et al., 2011). Agarwal et al. (2008) showedthat EMF emitted by mobile phones at an 850MHz frequency (max-imum power ≤ 1W, SAR 1.46W/kg) caused oxidative stress in semenand reduced spermatozoa motility and viability. Based on in vitro data,they concluded that carrying mobile phones in pockets causes dete-rioration in sperm quality due to exposure to oxidative stress (Agarwalet al., 2008a). Another retrospective study of 304 men by Wdowiaket al. (2007) observed a decline in reproductive age and a decrease insperm cell levels due to mobile phone use. In that study, while 65.7% ofpatients without mobile phones had prospective motile sperm ratesexceeding 50%, the figure was only 17% for subjects who had beenusing mobile phones for more than 2 years (Wdowiak et al., 2007).Recently, Oyewopo et al. (2017) concluded that impaired spermato-genic function may be associated with increased oxidative stress. Thisstress can be detected by elevated levels of malondialdehyde (MDA) inthe circulation after exposure to mobile phone-derived EMF-RF for2–3 h (Oyewopo et al., 2017). This finding supports the idea that in-creased MDA concentrations damage the cell plasma membrane byinducing lipid peroxidation in the sperm membrane, and that this thenresults in cell death. In this case, sperm motility and viability also re-duce the capacity of spermatozoa to undergo acrosomal reaction andfertilization (Eskiocak et al., 2006; Oyewopo et al., 2017).

2.2. The role of EMF in the apoptotic process underlying infertility

Male infertility is a growing problem worldwide. In this context, theassessment of possible side-effects of new technologies is a matter ofcritical importance (Yan et al., 2007). Because they play a key role insperm differentiation and maturation, alterations in the function ofcaspases can be an important contributor to or cause of infertility(Fig. 2). Caspase-3 is detected in both the perinuclear field and in thecytoplasm of germ cells. Conflicting studies exist with respect to therelationship between EMF and caspase. Some studies have shown thatexposure to EMF radiation for 45 days (1 h per day) does not suffi-ciently induce apoptosis in rat testis tissue (Paasch et al., 2004; Saidet al., 2004). Lee et al. (2010) performed a histological examination ofthe stages of spermatogenesis, germ cell numbers and apoptotic cellsemerging in the rat testis exposed to EMF at 848.5MHz (2.0W/kg) for12 weeks. Testicular levels of Bcl-2, p21, p53 and caspase-3 weremeasured. However, no significant effect on rat spermatogenesis wasobserved following subchronic exposure to 848.5-MHz EMF (Lee et al.,2010). Apoptosis plays a crucial role in regulating the numbers of germcells performing proliferation. However, some factors such as radiationexposure and H2O2 availability may increase the rate of apoptosis(Agarwal et al., 2011). In contrast to these negative studies, Kesari andBehari (2012) reported that exposure to RF radiation at 900MHz from amobile phone caused apoptosis leading to an increase in caspase-3 ac-tivity in sperm cells during spermiogenesis and sperm maturation, thusaffecting reproductive physiology (Kesari and Behari, 2012). Yilmaz

et al. (2008) examined Bcl-2 levels in the rat testis exposed to EMF for 1month (20min a day) and demonstrated using immunohistochemicalanalysis that mobile phones did not alter anti-apoptotic protein levels inthe rat testis (Yilmaz et al., 2008). Azadi Oskouyi et al. (2015) showedthat EMF induce apoptosis in epithelial cells and reduce the diameterand height of epithelial cells in the epididymis. This decrease may bedue to a reduction in metabolic activity in cells and in nuclear activity(Azadi Oskouyi et al., 2015).

2.3. The role of EMF in the hormonal regulation of spermatogenesis: cellularmechanisms

Several hormones are necessary for the regulation of the re-productive organs. Circulating hormones containing follicle-stimulatinghormone (FSH), luteinizing hormone (LH), activin B, inhibitor B, tes-tosterone and prolactin (PRL) regulate the first wave of spermatogen-esis (Barakat et al., 2008). In this context, Sepehrimanesh et al. (2014)investigated whether levels of serum testosterone, inhibin B, and pro-lactin, activin B, FSH and LH in adult male Sprague dawley rats wouldchange following continuous exposure to 900-MHz EMF. They reportedthat exposure to 900-MHz RF-EMF for 30 days increased activin B, FSH,LH, and PRL levels and reduced serum inhibin B and testosterone levelsin male rats. Although the physiological importance of these changes isstill unclear, the authors suggested that EMF may cause significantnegative effects on the male reproductive system (Sepehrimanesh et al.,2014). Further studies are needed to elucidate possible mechanisms.

Testosterone is a hormone primarily responsible for sperm produc-tion and growth. Testosterone synthesis is controlled by secretion of LHand FSH from the anterior pituitary gland in response to hypothalamicgonadotropin-releasing hormone (GnRH) release. Leydig cells induce ahigh intratesticular testosterone level that directly affects spermato-gonia and primary spermatocytes and promotes meiotic division. Theeffects of mobile phone EMF on testicular steroidogenesis have beenevaluated in this context (De Rosa et al., 2003). Wang et al. (2003)suggested that Leydig cells, which affect spermatogenesis, are the cellsthat are most sensitive to damage from EMF (Wang et al., 2003). Oxi-dative stress and alterations in the protein kinase enzyme complex inLeydig cells and seminiferous tubules may explain the EMF inducedimpairment in the functional structure of Leydig cells (Nikula et al.,1987). In addition, EMF has been shown not only to alter serum tes-tosterone, but also to affect P450scc mRNA expression in the testis insteroidogenesis (Zhou et al., 2005).

Forgacs et al. (2006) observed a decrease in serum testosterone levelbut no histopathological change in Leydig cells following 1800MHz ofGSM-like EMF. They concluded that Leydig cells are not the primarytarget of short-term exposure to EMF and that additional study wasrequired to determine factors affecting testosterone production(Forgacs et al., 2006). Salama et al. (2009) examined androgen-de-pendent secretory activity in rabbits exposed to EMF and detected adose-related decrease in seminal plasma fructose. However, no changewas observed in serum testosterone levels in the EMF-exposed groups.Changes in testosterone receptors and enhanced oxidative stress in ac-cessory glands have also been reported to occur in human males(Salama et al., 2009). In contrast, others report that short-term EMFexposures have no observable effect on the pituitary gland or the pro-duction of gonadotropins both humans and animals exposed to EMF.Thus, De Seze et al. (1998) evaluated the concentrations of gonado-tropin in 21 healthy men exposed to 900MHz RF for 1 month (2 h perday, 5 days weekly) and observed no effect (de Seze et al., 1998).

Chronic exposure to the EMF emitted from mobile phones causesdeteriorations in testicular functions and decreased gonadotropic hor-mone levels linked with increased oxidative stress (Oyewopo et al.,2017). Gutschi et al. (2011) investigated 2110 men attending an Aus-trian infertility clinic between 1993 and 2007 and evaluated the serumlevels of fertility-related hormones (FSH, LH, testosterone, and PRL).They concluded that mobile phones affect sperm quality and impair

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male fertility (Gutschi et al., 2011). Another study investigated theeffects of EMF exposure on plasma hormonal biomarkers and observeda lower testosterone level in the group exposed to EMF compared to thecontrol group. The authors concluded that chronic EMF exposure mayreduce levels of plasma testosterone and the ratio of testosterone/es-tradiol in males (Wang et al., 2016). Many animal studies have alsoreported that exposure to EMF can reduce serum or plasma testosterone(Kesari and Behari, 2012; Ozguner et al., 2005; Shahin et al., 2014). Forinstance, decreased testosterone levels may lead to EMF-induced da-mage in Leydig cells (Sepehrimanesh et al., 2014).

In addition to a number of studies showing that EMF may haveharmful effects on sperm function, there have also been many reportsindicating that such radiation can also affect the testicles. Exposure ofmale rats to EMF twice daily for 60min may lead to dilation of semi-niferous tubules (Al-Damegh, 2012). Dasdag et al. (1999) reportedthinning of the seminiferous tubules as a reaction to exposure to mobilephone for 1 month (2 h a day). In a subsequent study, they also reportedthat daily exposure to 20-min EMF for 1 month does not result in anychanges in testicular structure (Dasdag et al., 2003). In addition to thepotential effects on the diameter of seminiferous tubules, chronic ex-posure (3 h a day for a year) to EMF causes a decrease in tunica albu-ginea thickness in the rat testis (Tas et al., 2014).

3. The metabolomic effects of EMF on the female reproductivesystem

The female reproductive organs have critical functions for the sur-vival of the species. Damage to female reproductive tissue or processesfrom both low frequency EMF as well as higher frequency RF due to thewidespread use of mobile phones (Naziroglu et al., 2013) may increasethe risk for infertility or contribute to abnormal fetal growth (Fig. 4)(Poulletier de Gannes et al., 2013). Data obtained from both animal andhuman studies of EMF have shown adverse effects on granulosa cells,numbers of ovarian follicles, endometrial tissue, oocyte and embryoquality, and even changes in fetal heart physiology during pregnancy(Batellier et al., 2008; Diem et al., 2005; Merhi, 2012). Male offspringexposed prenatally to EMF develop a range of deficiencies in Leydigcells as well as reduced levels of testosterone and have also been

associated with increased behavioral anomalies (Aldad et al., 2012).

3.1. Morphological alterations in female infertility caused by mobile phoneuse

A considerable number of studies have investigated the impacts ofEMF on the ovaries. Roushangar and Rad (2007) studied the effect oflow frequency EMF on follicle development in the rat ovary. Their re-sults indicated thinner nuclei of oocytes and zona pellucida in an EMF-exposed group compared to a matched control group. Apoptotic bodieswere more common in EMF-exposed rats than controls (Roushangarand Rad, 2007). Morphological alterations observed in the oocytes in-dicate the cytotoxic effect of EMF. Researchers have suggested that EMFexposure may disturb normal folliculogenesis. Roshanger et al. (2014)studied the effects of exposure to low frequency EMF on oocyte dif-ferentiation and follicle growth. They concluded that EMF exposureduring the growth period may affect both oocyte differentiation andfolliculogenesis, and that it may decrease fertility by reducing ovaryreservoirs (Roshangar et al., 2014). Türedi et al. (2016) reported de-generation in granulosa cells and vacuolization of granulosa cells to-wards the antrum of ovarian tissue in rats exposed to 900MHz EMFduring the prenatal period (Turedi et al., 2016). Similarly, severalstudies have indicated that histo-morphological evidence of the apop-totic process in the granulosa cells and the ovary is supported by de-generative findings, such as chromatin changes, cytoplasmic vacuoli-zation, inflation, nuclear concentration and nuclear destruction (Booneet al., 1997; Hughes and Gorospe, 1991). Oral et al. (2006) examinedoxidative stress and the apoptotic process in endometrial tissue fol-lowing the RF-EMF exposure for 30 days (30min per day). They usedimmunohistochemical methods to investigate Bcl-2, caspase-3, caspase-8, Bax and MDA in order to evaluate the lipid peroxidation as an in-dicator of endometrial disorder induced by oxidative stress. The authorsunderlined that 900MHz EMF caused oxidative stress and endometrialapoptosis (Oral et al., 2006).

Sangun et al. (2015) investigated the effects of long-term 2450MHzEMF exposure on pubertal development and growth of female Wistaralbino rats. They concluded that prenatal exposure to 2450MHz EMFcaused delayed puberty and restriction in postnatal growth (Sangunet al., 2015). In contrast, Poulletier de Gannes et al. (2013) reportedthat exposure to Wi-Fi for 1 h per day (6 days/week) for 3 weeks ofgestation had no gross adverse effects on the reproductive organs andfertility in either male or female rats (Poulletier de Gannes et al., 2013).Oral et al. (2006) suggested that exposure to 900MHz EMF has a sig-nificant effect on the rat endometrium and that the production of ROS isrelated to experimental conditions. In that context, an increase in theMDA levels as an indicator in the increased ROS production has beenreported. An association between the lipid peroxidation and EMF-in-duced damage may reflect the pathological mechanism resulting fromexposure (Oral et al., 2006). For these as with all studies the lack ofstandardization of exposure metrics hampers the interpretation of thefindings.

3.2. The effects of EMF exposure on oogenesis

Margaritis et al. (2014) evaluated the effects of exposure to GSM-like 900MHz EMF on apoptosis of the follicles in oogenesis in Droso-phila virilis and Drosophila melanogaster. Their findings revealed that 900and 1800MHz EMF led to a significant reduction in fertility in D. virilis.A decrease of 30% was observed in the group exposed to 900MHz EMF,while a decrease of 20% was observed in the group exposed to 900MHzEMF (Margaritis et al., 2014). Notably, exposure to the EMF emitted byGSM for 2min/a day causes a decrease in the reproductive capacity ofD. melanogaster during adulthood (Panagopoulos et al., 2004). Simi-larly, Panagopoulos (2012) investigated D. melanogaster exposed toGSM radiation and reported that mobile phones caused delayed ovariandevelopment because of DNA damage resulting in apoptosis in the

Fig. 4. The effect of EMF on female reproduction and fetal development. IUGR:Intrauterine growth restriction (Agarwal et al., 2012).

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ovary. There are some similarities in EMF-induced fertility disordersbetween insects and mammals in terms of induction of apoptosis(Panagopoulos, 2012). Previous studies have shown that antioxidantbiochemical changes may occur due to long-term EMF exposure(Agarwal et al., 2009). Yüksel et al. (2016) investigated the effects of900, 1800 and 2450MHz EMF emitted by mobile phones and Wi-Fi onthe uterus in pregnant rats and their offspring by evaluating levels of

plasma hormones and the oxidative stress parameters (Yuksel et al.,2016). Additionally, EMF-induced damage may occur through theconsumption of non-enzymatic antioxidants and enzymatic anti-oxidants (Naziroglu et al., 2013; Ozorak et al., 2013). GSH-Px is thethiol antioxidant enzyme in mammalian cells and protects cytosoliclipids, proteins and nucleic acids as a response to oxidative damage(Anderson, 1998; Naziroglu, 2009). Yüksel et al. (2016) observed de-creased levels of prolactin, estrogen and progesterone in pregnants ratsexposed to 900, 1800 and 2450 MHz EMF and their offspring. They alsonoted that lipid peroxidation in the rat uterus, was linked with a declinein GSH-Px, the key antioxidant enzyme, in EMF. However, no changeswere observed in the rat developmental period. These findings suggestthat the antioxidant response of GSH can be upregulated through theenzymatic activity of GSH-Px (Fig. 5) (Yuksel et al., 2016).

4. The effects of mobile phone use on fertilization and pregnancy

Reproductive cells seem to be more sensitive to both ionizing andnon-ionizing radiation than other cells because they undergo rapid ratesof growth during meiosis and mitosis. The faster that cells grow thegreater their chance of incorporating errors during the synthesis ofvarious biomolecules (Panagopoulos, 2012). EMF induces the genera-tion of ROS in various animal and human tissues (Kismali et al., 2012;Ozorak et al., 2013; Seite et al., 2004).

When the water present in approximately 70–80% of cells is ex-posed to EMF, decomposition can occur. The body's electrical con-ductivity increases due to increased water consumption, and amnioticfluids during pregnancy can also result in greater sensitivity to EMF.(Kismali et al., 2012; Obolenskaya et al., 2010).

Chen et al. (2017) studied the effects of EMF emitted by simulatedmobile phone use on fertilization and embryo development. They re-ported that 935MHz EMF in medium and high-density can reducefertilization rates and blastulation in mice, thus lowering the prob-ability of embryo implantation. Alanine, glycine, glutamic acid andglutamine promote embryo development during the cleavage stages(Chen et al., 2017). Heynick and Merritt reported that RF may interactwith the embryo or fetus during pregnancy and cause developmental

Fig. 5. The role of TRPV1-mediated Ca+2 channels in oxidative stress -induced damage inthe rat uterus exposed to long-term EMF during pregnancy. The figure also shows theproduction of superoxide (O2

−) and hydrogen peroxide (H2O2). Glutathione peroxidase(GSH-Px) catalyzes the reduction of H2O2 to H2O (modified from Yuksel et al., 2016).

Table 1Summary of the metabolomics effects of mobile phones on reproductivity.

Frequency Study type Dose/time Conclusion/mechanism References

900MHz EMF-induced oxidative stress increased in the development of offspring whenthe maternal uterus was exposed tho EMF.1800MHz Rats 60min/day (Yuksel et al., 2016)

2450MHz2450MHz Rats 60 days daily

2 h2450MHz EMF exposure leads to apoptosis by increasing caspase-3 activityin the rat sperm during spermiogenesis and affects sperm maturation

(Kumar et al., 2011)

850MHz Human 60min/day RF-EMF emitted by mobile phones may cause oxidative stress in humansemen and affect sperm mobility and viability.

(Agarwal et al., 2009)

900MHz Rats 35 days Following EMF exposure, an increase in CAT activity and a decrease in GPx,SOD activity may cause oxidative stress and enhance the possibility ofinfertility.

(Kesari et al., 2011)Daily 2 h

900MHz Rats 1 month No effect was observed in the testis following 20min exposure to EMFemitted by mobile phones.

(Dasdag et al., 2003)7days/week20min/day

1900MHz Rats 18 week Long-term exposure to EMF may cause a decrease in sperm motility. (Kang et al., 2010)(800MHz digital and

800MHz analog)6 h/day

900MHz Rats 4 weeks Long-term EMF exposure may affect the hormonal regulation ofspermiogenesis

(Ozguner et al., 2005)5 days /week30min/day

800MHz Rabbits 12 weeks When mobile phones are kept in standby mode, EMF may causedeteriorations in testicular morphology and functions in the adult rabbit.

(Salama et al., 2010)8 h/day

900/ 1800MHz Rats 28 days Exposure to RF-EMF leads to an increase in lipid peroxidation and low GSHlevels in the epididymis and testis.

(Mailankot et al., 2009)1 h/day

900MHz Drosophilamelanogaster

5 days Exposure to continuous 900MHz EMF leads to actin-cytoskeletondisorganization resulting in cell death during early and mid-oogenesis.

(Panagopoulos et al.,2004)6min/day

2450MHz Rats 21 days EMF exposure caused an increase in total antioxidant status and oxidativestress index. More apoptotic cells were observed in prenatal rats exposed toEMF.

(Sangun et al., 2015)1 h/day

G. Altun et al. Environmental Research 167 (2018) 700–707

705

abnormalities. It has also been suggested that EMF exposure can resultin fetal death or mutations (Heynick and Merritt, 2003) along withattention deficit disorders in offspring (Aldad et al., 2012).

In conclusion, EMF emitted by mobile phones has a number of well-documented adverse metabolomic effects on the male and female re-productive systems and can lead to infertility by increasing ROS pro-duction and reducing GSH and other antioxidants. The primary targetof the EMF emitted by mobile phones may be the cell membrane (Pall inpress, this volume). This then results in accelerated activity of mem-brane NADH oxidase and, consequently, greater rates of ROS formationthat cannot be easily conjugated or detoxified. Although many studieshave reported morphological and functional deteriorations in testis andovary following EMF exposures, as well both structural and functionaldeficits in reproductive health, the underlying mechanisms have notbeen fully elucidated. To assist in further clarification of these processesand mechanisms, Table 1 summarizes key studies on the metabolomiceffects of EMF on reproductive systems. Future studies will benefitgreatly from standardized exposure protocols and evaluations of keymetabolomic indicators.

Conflict the interest

The authors declare that they have no conflict of interest.

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